Targeting of Tsc13p to nucleus-vacuole junctions: a role for very-long-chain fatty acids in the biogenesis of microautophagic vesicles

TSC13 is required for the biosynthesis of very-long-chain fatty acids (VLCFAs) in yeast. Tsc13p is a polytopic endoplasmic reticulum (ER) membrane protein that accumulates at nucleus-vacuole (NV) junctions, which are formed through Velcro-like interactions between Nvj1p in the perinuclear ER and Vac8p on the vacuole membrane. NV junctions mediate piecemeal microautophagy of the nucleus (PMN), during which bleb-like portions of the nucleus are extruded into invaginations of the vacuole membrane and degraded in the vacuole lumen. We report that Tsc13p is sequestered into NV junctions from the peripheral ER through Vac8p-independent interactions with Nvj1p. During nutrient limitation, Tsc13p is incorporated into PMN vesicles in an Nvj1p-dependent manner. The lumenal diameters of PMN blebs and vesicles are significantly reduced in tsc13-1 and tsc13-1 elo3-Delta mutant cells. PMN structures are also smaller in cells treated with cerulenin, an inhibitor of de novo fatty acid synthesis and elongation. The targeting of Tsc13p-GFP into NV junctions is perturbed by cerulenin, suggesting that its binding to Nvj1p depends on the availability of fatty acid substrates. These results indicate that Nvj1p retains and compartmentalizes Tsc13p at NV junctions and that VLCFAs contribute to the normal biogenesis of trilaminar PMN structures in yeast.     

Nucleus-vacuole junctions and piecemeal microautophagy of the nucleus in S. cerevisiae

Various modes of autophagy conspire to degrade virtually every compartment of the eukaryotic cell. In Saccharomyces cerevisiae, a process called "piecemeal microautophagy of the nucleus" (PMN) even pinches off and degrades nonessential portions of the nucleus. PMN is a constitutive process induced to high levels by starvation or rapamycin, an inhibitor of TOR kinase. PMN occurs at nucleus-vacuole (NV) junctions, which are Velcro-like patches formed by interactions between the vacuole membrane protein Vac8p and the outer-nuclear-membrane protein Nvj1p. In response to nutrient depletion, Nvj1p increasingly binds and sequesters two proteins with roles in lipid metabolism, Osh1p and Tsc13p. Tsc13p is required for the normal biogenesis of PMN vesicles. The sequestration of Osh1p by Nvj1p likely serves to negatively regulate the trafficking of tryptophan permease(s) to the plasma membrane. Thus, NV junctions and PMN orchestrate novel and sophisticated responses to nutrient limitation.     

The luminal N-terminus of yeast Nvj1 is an inner nuclear membrane anchor

The endoplasmic reticulum (ER) in Saccharomyces cerevisiae is largely divided between perinuclear and cortical compartments. Yeast Nvj1 localizes exclusively to small patches on the perinuclear ER where it interacts with Vac8 in the vacuole membrane to form nucleus-vacuole (NV) junctions. Three regions of Nvj1 mediate the biogenesis of NV junctions. A membrane-spanning domain targets the protein to the ER. The C-terminus binds Vac8 in the vacuole membrane, which induces the clustering of both proteins into NV junctions. The luminal N-terminus is required for strict perinuclear localization. Three-dimensional cryo-electron tomography reveals that Nvj1 clamps the separation between the two nuclear membranes to half the width of bulk nuclear envelope. The N-terminus contains a hydrophobic sequence bracketed by basic residues that resembles outer mitochondrial membrane signal-anchors. The hydrophobic sequence can be scrambled or reversed without affecting function. Mutations that reduce the hydrophobicity of the core sequence or affect the distribution of basic residues cause mislocalization to the cortical ER. We conclude that the N-terminus of Nvj1 is a retention sequence that bridges the perinuclear lumen and inserts into the inner nuclear membrane.     

Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae

Nucleus-vacuole (NV) junctions in Saccharomyces cerevisiae are formed through specific interactions between Vac8p on the vacuole membrane and Nvj1p in the nuclear envelope. Herein, we report that NV junctions in yeast promote piecemeal microautophagy of the nucleus (PMN). During PMN, teardrop-like blebs are pinched from the nucleus, released into the vacuole lumen, and degraded by soluble hydrolases. PMN occurs in rapidly dividing cells but is induced to higher levels by carbon and nitrogen starvation and is under the control of the Tor kinase nutrient-sensing pathway. Confocal and biochemical assays demonstrate that Nvj1p is degraded in a PMN-dependent manner. PMN occurs normally in apg7-delta cells and is, therefore, not dependent on macroautophagy. Transmission electron microscopy reveals that portions of the granular nucleolus are often sequestered into PMN structures. These results introduce a novel mode of selective microautophagy that targets nonessential components of the yeast nucleus for degradation and recycling in the vacuole.     

Microautophagy of the nucleus coincides with a vacuolar diffusion barrier at nuclear-vacuolar junctions

Nuclei bind yeast vacuoles via nucleus-vacuole (NV) junctions. Under nutrient restriction, NV junctions invaginate and release vesicles filled with nuclear material into vacuoles, resulting in piecemeal microautophagy of the nucleus (PMN). We show that the electrochemical gradient across the vacuolar membrane promotes invagination of NV junctions. Existing invaginations persist independently of the gradient, but final release of PMN vesicles requires again V-ATPase activity. We find that NV junctions form a diffusion barrier on the vacuolar membrane that excludes V-ATPase but is enriched in the VTC complex and accessible to other membrane-integral proteins. V-ATPase exclusion depends on the NV junction proteins Nvj1p,Vac8p, and the electrochemical gradient. It also depends on factors of lipid metabolism, such as the oxysterol binding protein Osh1p and the enoyl-CoA reductase Tsc13p, which are enriched in NV junctions, and on Lag1p and Fen1p. Our observations suggest that NV junctions form in two separable steps: Nvj1p and Vac8p suffice to establish contact between the two membranes. The electrochemical potential and lipid-modifying enzymes are needed to establish the vacuolar diffusion barrier, invaginate NV junctions, and form PMN vesicles.     

Piecemeal microautophagy of the nucleus: Genetic and morphological traits

Nucleus-vacuole (NV) junctions are formed in Saccharomyces cerevisiae through interactions between Vac8 in the vacuole membrane and Nvj1 in the perinuclear ER. Upon starvation, vesicles containing part of the nucleus emanate from these contact sites and finally pinch off into invaginations of the vacuole. Due to its morphological similarity to microautophagy this process had been termed "piecemeal microautophagy of the nucleus" (PMN). We recently discovered that a number of ATG genes required for macroautophagy and micropexophagy are also required for PMN and accordingly named it micronucleophagy. Therefore, PMN represents a novel model system to investigate the functions of the highly conserved but poorly understood core autophagic apparatus. We here extend the morphological analysis of PMN using immunogold and freeze fracture electron microscopy.     

Vac8p release from the SNARE complex and its palmitoylation are coupled and essential for vacuole fusion

Activated fatty acids stimulate budding and fusion in several cell-free assays for vesicular transport. This stimulation is thought to be due to protein palmitoylation, but relevant substrates have not yet been identified. We now report that Vac8p, a protein known to be required for vacuole inheritance, becomes palmitoylated when isolated yeast vacuoles are incubated under conditions that allow membrane fusion. Similar requirements for Vac8p palmitoylation and vacuole fusion, the inhibition of vacuole fusion by antibodies to Vac8p and the strongly reduced fusion of vacuoles lacking Vac8p suggest that palmitoylated Vac8p is essential for homotypic vacuole fusion. Strikingly, palmitoylation of Vac8p is blocked by the addition of antibodies to Sec18p (yeast NSF) only. Consistent with this, a portion of Vac8p is associated with the SNARE complex on vacuoles, which is lost during Sec18p- and ATP-dependent priming. During or after SNARE complex disassembly, palmitoylation occurs and anchors Vac8p to the vacuolar membrane. We propose that palmitoylation of Vac8p is regulated by the same machinery that controls membrane fusion.     

Vac8p, a Vacuolar Protein with Armadillo Repeats, Functions in both Vacuole Inheritance and Protein Targeting from the Cytoplasm to Vacuole

During each cell cycle, the yeast vacuole actively partitions between mother and daughter cells. This process requires actin, profilin, an unconventional myosin (Myo2p), and Vac8p. A mutant yeast strain, vac8, is defective in vacuole inheritance, specifically, in early vacuole migration. Vac8p is a 64-kD protein found on the vacuole membrane, a site consistent with its role in vacuole inheritance. Both myristoylation and palmitoylation are required for complete Vac8p localization. Interestingly, whereas myristoylation of Vac8p is not required for vacuole inheritance, palmitoylation is essential. Thus, palmitoylation appears to play a more direct role in vacuole inheritance. Most of the VAC8 sequence encodes 11 armadillo (Arm) repeats. Arm repeats are thought to mediate protein–protein interactions, and many Arm proteins have multiple functions. This is also true for Vac8p. In addition to its role in early vacuole inheritance, Vac8p is required to target aminopeptidase I from the cytoplasm to the vacuole. Mutant analysis demonstrates that Vac8p functions separately in these two processes. Vac8p cosediments with actin filaments. Vac8p is related to β-catenin and plakoglobin, which connect a specific region of the plasma membrane to the actin cytoskeleton. In analogy, Vac8p may link the vacuole to actin during vacuole partitioning.     

Palmitoylation plays a role in targeting Vac8p to specific membrane subdomains

Vac8p is a multifunctional yeast protein involved in several distinct vacuolar events including vacuole inheritance, vacuole homotypic fusion, nucleus-vacuole junction formation and the cytoplasm to vacuole protein targeting pathway. Vac8p associates with the vacuole membrane via myristoylation and palmitoylation. Vac8p has three putative palmitoylation sites, at Cys 4, 5 and 7. Here, we show that each of these cysteines may serve as a palmitoylation site. Palmitoylation at Cys 7 alone provides partial function of Vac8p, whereas palmitoylation at either Cys 4 or Cys 5 alone is sufficient for Vac8p function. In the former mutant, there is a severe defect in the localization of Vac8p to the vacuole membrane, while in the latter mutants, there is a partial defect in the localization of Vac8p. In addition, our studies provide evidence that palmitoylation targets Vac8p to specific membrane subdomains.     

The vacuolar DHHC-CRD protein Pfa3p is a protein acyltransferase for Vac8p

Palmitoylation of the vacuolar membrane protein Vac8p is essential for vacuole fusion in yeast (Veit, M., R. Laage, L. Dietrich, L. Wang, and C. Ungermann. 2001. EMBO J. 20:3145-3155; Wang, Y.X., E.J. Kauffman, J.E. Duex, and L.S. Weisman. 2001. J. Biol. Chem. 276:35133-35140). Proteins that contain an Asp-His-His-Cys (DHHC)-cysteine rich domain (CRD) are emerging as a family of protein acyltransferases, and are therefore candidates for mediators of Vac8p palmitoylation. Here we demonstrate that the DHHC-CRD proteins Pfa3p (protein fatty acyltransferase 3, encoded by YNL326c) and Swf1p are important for vacuole fusion. Cells lacking Pfa3p had fragmented vacuoles when stressed, and cells lacking both Pfa3p and Swf1p had fragmented vacuoles under normal growth conditions. Pfa3p promoted Vac8p membrane association and palmitoylation in vivo and partially purified Pfa3p palmitoylated Vac8p in vitro, establishing Vac8p as a substrate for palmitoylation by Pfa3p. Vac8p is the first N-myristoylated, palmitoylated protein identified as a substrate for a DHHC-CRD protein.     

Dynamic localization of yeast Fus2p to an expanding ring at the cell fusion junction during mating

Fus2p is a pheromone-induced protein associated with the amphiphysin homologue Rvs161p, which is required for cell fusion during mating in Saccharomyces cerevisiae. We constructed a functional Fus2p-green fluorescent protein (GFP), which exhibits highly dynamic localization patterns in pheromone-responding cells (shmoos): diffuse nuclear, mobile cytoplasmic dots and stable cortical patches concentrated at the shmoo tip. In mitotic cells, Fus2p-GFP is nuclear but becomes cytoplasmic as cells form shmoos, dependent on the Fus3p protein kinase and high levels of pheromone signaling. The rapid cytoplasmic movement of Fus2p-GFP dots requires Rvs161p and polymerized actin and is aberrant in mutants with compromised actin organization, which suggests that the Fus2p dots are transported along actin cables, possibly in association with vesicles. Maintenance of Fus2p-GFP patches at the shmoo tip cortex is jointly dependent on actin and a membrane protein, Fus1p, which suggests that Fus1p is an anchor for Fus2p. In zygotes, Fus2p-GFP forms a dilating ring at the cell junction, returning to the nucleus at the completion of cell fusion.     

Fusion of docked membranes requires the armadillo repeat protein Vac8p.

The discovery of molecules required for membrane fusion has revealed a remarkably conserved mechanism that centers upon the formation of a complex of SNARE proteins. However, whether the SNARE proteins or other components catalyze the final steps of membrane fusion in vivo remains unclear. Understanding this last step depends on the identification of molecules that act late in the fusion process. Here we demonstrate that in Saccharomyces cerevisiae, Vac8p, a myristoylated and palmitoylated armadillo repeat protein, is required for homotypic vacuole fusion. Vac8p is palmitoylated during the fusion reaction, and the ability of Vac8p to be palmitoylated appears to be necessary for its function in fusion. Both in vivo and in vitro analyses show that Vac8p functions after both Rab-dependent vacuole docking and the formation of trans-SNARE pairs. We propose that Vac8p may bind the fusion machinery through its armadillo repeats and that palmitoylation brings this machinery to a specialized lipid domain that facilitates bilayer mixing.     

A novel cold-sensitive allele of the rate-limiting enzyme of fatty acid synthesis, acetyl coenzyme A carboxylase, affects the morphology of the yeast vacuole through acylation of Vac8p

The yeast vacuole functions both as a degradative organelle and as a storage depot for small molecules and ions. Vacuoles are dynamic reticular structures that appear to alternately fuse and fragment as a function of growth stage and environment. Vac8p, an armadillo repeat-containing protein, has previously been shown to function both in vacuolar inheritance and in protein targeting from the cytoplasm to the vacuole. Both myristoylation and palmitoylation of Vac8p are required for its efficient localization to the vacuolar membrane (Y.-X. Wang, N. L. Catlett, and L. S. Weisman, J. Cell Biol. 140:1063-1074, 1998). We report that mutants with conditional defects in the rate-limiting enzyme of fatty acid synthesis, acetyl coenzyme A carboxylase (ACC1), display unusually multilobed vacuoles, similar to those observed in vac8 mutant cells. This vacuolar phenotype of acc1 mutant cells was shown biochemically to be accompanied by a reduced acylation of Vac8p which was alleviated by fatty acid supplementation. Consistent with the proposed defect of acc1 mutant cells in acylation of Vac8p, vacuolar membrane localization of Vac8p was impaired upon shifting acc1 mutant cells to nonpermissive condition. The function of Vac8p in protein targeting, on the other hand, was not affected under these conditions. These observations link fatty acid synthesis and availability to direct morphological alterations of an organellar membrane.     

Vac8p, an armadillo repeat protein, coordinates vacuole inheritance with multiple vacuolar processes

Vac8p, an armadillo (ARM) repeat protein, is required for multiple vacuolar processes. It functions in vacuole inheritance, cytoplasm-to-vacuole protein targeting pathway, formation of the nucleus-vacuole junction and vacuole-vacuole fusion. These functions each utilize a distinct Vac8p-binding partner. Here, we report an additional Vac8p function: caffeine resistance. We show that Vac8p function in caffeine resistance is mediated via a newly identified Vac8p-binding partner, Tco89p. The interaction between Vac8p and each binding partner requires an overlapping subset of Vac8p ARM repeats. Moreover, these partners can compete with each other for access to Vac8p. Furthermore, Vac8p is enriched in three separate subdomains on the vacuole, each with a unique binding partner dedicated to a different vacuolar function. These findings suggest that a major role of Vac8p is to spatially separate multiple functions thereby enabling vacuole inheritance to occur concurrently with other vacuolar processes.     

Phr1p, a glycosylphosphatidylinsitol-anchored β(1,3)-glucanosyltransferase critical for hyphal wall formation, localizes to the apical growth sites and septa in Candida albicans

Cell wall biogenesis is a dynamic process relying on the coordinated activity of several extracellular enzymes. PHR1 is a pH-regulated gene of Candida albicans encoding a glycosylphosphatidylinositol-anchored β(1,3)-glucanosyltransferase of family GH72 which acts as a cell wall remodelling enzyme and is crucial for morphogenesis and virulence. In order to explore the function of Phr1p, we obtained a green fluorescent protein (GFP) fusion to determine its localization. During induction of vegetative growth, Phr1p-GFP was concentrated in the plasma membrane of the growing bud, in the mother-bud neck, and in the septum. Phr1p-GFP was recovered in the detergent-resistant membranes indicating its association with the lipid rafts as the wild type Phr1p. Upon induction of hyphal growth, Phr1p-GFP highly concentrated at the apex of the germ tubes and progressively distributed along the lateral sides of the hyphae. Phr1p-GFP also labelled the hyphal septa, where it colocalized with chitin. Localization to the hyphal septa was perturbed in nocodazole-treated cells, whereas inhibition of actin polymerization hindered the apical localization. Electron Microscopy analysis of the hyphal wall ultrastructure of a PHR1 null mutant showed loss of compactness and irregular organization of the surface layer. These observations indicate that Phr1p plays a crucial role in hyphal wall formation, a highly regulated process on which morphogenesis and virulence rely.     

Atg18 regulates organelle morphology and Fab1 kinase activity independent of its membrane recruitment by phosphatidylinositol 3,5-bisphosphate

The lipid kinase Fab1 governs yeast vacuole homeostasis by generating PtdIns(3,5)P(2) on the vacuolar membrane. Recruitment of effector proteins by the phospholipid ensures precise regulation of vacuole morphology and function. Cells lacking the effector Atg18p have enlarged vacuoles and high PtdIns(3,5)P(2) levels. Although Atg18 colocalizes with Fab1p, it likely does not directly interact with Fab1p, as deletion of either kinase activator-VAC7 or VAC14-is epistatic to atg18Delta: atg18Deltavac7Delta cells have no detectable PtdIns(3,5)P(2). Moreover, a 2xAtg18 (tandem fusion) construct localizes to the vacuole membrane in the absence of PtdIns(3,5)P(2), but requires Vac7p for recruitment. Like the endosomal PtdIns(3)P effector EEA1, Atg18 membrane binding may require a protein component. When the lipid requirement is bypassed by fusing Atg18 to ALP, a vacuolar transmembrane protein, vac14Delta vacuoles regain normal morphology. Rescue is independent of PtdIns(3,5)P(2), as mutation of the phospholipid-binding site in Atg18 does not prevent vacuole fission and properly regulates Fab1p activity. Finally, the vacuole-specific type-V myosin adapter Vac17p interacts with Atg18p, perhaps mediating cytoskeletal attachment during retrograde transport. Atg18p is likely a PtdIns(3,5)P(2)"sensor," acting as an effector to remodel membranes as well as regulating its synthesis via feedback that might involve Vac7p.     

ATP-independent control of Vac8 palmitoylation by a SNARE subcomplex on yeast vacuoles

Yeast vacuole fusion requires palmitoylated Vac8. We previously showed that Vac8 acylation occurs early in the fusion reaction, is blocked by antibodies against Sec18 (yeast N-ethylmaleimide-sensitive fusion protein (NSF)), and is mediated by the R-SNARE Ykt6. Here we analyzed the regulation of this reaction on purified vacuoles. We show that Vac8 acylation is restricted to a narrow time window, is independent of ATP hydrolysis by Sec18, and is stimulated by the ion chelator EDTA. Analysis of vacuole protein complexes indicated that Ykt6 is part of a complex distinct from the second R-SNARE, Nyv1. We speculate that during vacuole fusion, Nyv1 is the classical R-SNARE, whereas the Ykt6-containing complex has a novel function in Vac8 palmitoylation.     

Vacuole size control: regulation of PtdIns(3,5)P2 levels by the vacuole-associated Vac14-Fig4 complex, a PtdIns(3,5)P2-specific phosphatase

In the budding yeast Saccharomyces cerevisiae, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is synthesized by a single phosphatidylinositol 3-phosphate 5-kinase, Fab1. Cells deficient in PtdIns(3,5)P2 synthesis exhibit a grossly enlarged vacuole morphology, whereas increased levels of PtdIns(3,5)P2 provokes the formation of multiple small vacuoles, suggesting a specific role for PtdIns(3,5)P2 in vacuole size control. Genetic studies have indicated that Fab1 kinase is positively regulated by Vac7 and Vac14; deletion of either gene results in ablation of PtdIns(3,5)P2 synthesis and the formation of a grossly enlarged vacuole. More recently, a suppressor of vac7Delta mutants was identified and shown to encode a putative phosphoinositide phosphatase, Fig4. We demonstrate that Fig4 is a magnesium-activated PtdIns(3,5)P2-selective phosphoinositide phosphatase in vitro. Analysis of a Fig4-GFP fusion protein revealed that the Fig4 phosphatase is localized to the limiting membrane of the vacuole. Surprisingly, in the absence of Vac14, Fig4-GFP no longer localizes to the vacuole. However, Fig4-GFP remains localized to the grossly enlarged vacuoles of vac7 deletion mutants. Consistent with these observations, we found that Fig4 physically associates with Vac14 in a common membrane-associated complex. Our studies indicate that Vac14 both positively regulates Fab1 kinase activity and directs the localization/activation of the Fig4 PtdIns(3,5)P2 phosphatase.     

Early and late molecular events of glucose-induced pexophagy in Pichia pastoris require Vac8

We have identified the Pichia pastoris Vac8 homolog, a 60-64 kDa armadillo repeat protein, and have examined the role of PpVac8 in the degradative pathways involving the yeast vacuole. We report here that PpVac8 is required for glucose-induced pexophagy, but not ethanol-induced pexophagy or starvation-induced autophagy. This has been demonstrated by the persistence of peroxisomal alcohol oxidase activity in mutants lacking PpVac8 during glucose adaptation. During glucose-induced micropexophagy, in the absence of PpVac8, the vacuole was invaginated with arm-like "segmented" extensions that almost completely surrounded the adjacent peroxisomes. Vac8-GFP was found at the vacuolar membrane and concentrated at the base of the arm-like protrusions that extend from the vacuole to sequester the peroxisomes. The localization of Vac8-GFP to the vacuolar membrane occurred independent of PpAtg1, PpAtg9 or PpAtg11. Mutagenesis of the palmitoylated cysteines to alanines or deletion of the myristoylation and palmitoylation sites of PpVac8 resulted in decreased protein stability, impaired vacuolar association and reduced degradation of peroxisomal alcohol oxidase. Deletion of the central armadillo repeat domains of the PpVac8 did not alter its association with the vacuolar membrane, but resulted in a non-functional protein that suppressed the formation of the arm-like extensions from the vacuole to engulf the peroxisomes. PpVac8 is essential for the trafficking of PpAtg11, but not PpAtg1 or PpAtg18, to the vacuole membrane. Together, our results support a role for PpVac8 in early (formation of sequestering membranes) and late (post-MIPA membrane fusion) molecular events of glucose-induced pexophagy.     

Role of Vac8 in Formation of the Vacuolar Sequestering Membrane during Micropexophagy

Vac8 is a yeast vacuolar membrane protein involved in vacuolar membrane dynamics, e.g., vacuole inheritance and vacuolar membrane fusion. This protein is also necessary for a subset of autophagic pathways that deliver specific cellular components to the vacuole. In this study, we show that the micropexohagy and vacuole inheritance required distinct domain structures of Pichia pastoris Vac8 (PpVac8). Whereas vacuole inheritance required the Armadillo repeat (ARM) region that resides in the middle part of the protein, micropexophagy did not. Deletion of both the ARM and C-terminal domains inhibited a characteristic of vacuolar dynamics during micropexophagy, i.e., formation of the vacuolar sequestering membrane (VSM). Subsequent analyses indicated that PpVAC8 disruption abolished recruitment of PpAtg11, another protein required for formation of the VSM, to the vacuolar membrane. These results present a novel molecular function of PpVac8 in micropexophagy.